The long range goal of this research is to elucidate the molecular mechanisms involved in the regulation of hormone synthesis and secretion by anterior pituitary cells. The proposed studies are designed to investigate how hormones, particularly the hypothalamic tripeptide thyrotropin- releasing hormone (TRH) interact with their receptors and transmit their signals. TRH is believed to interact with a membrane receptor and activate phospholipid turnover through a G-protein leading to an increase in inositol triphosphate, which stimulates calcium release from intracellular stores, and in diacylglycerol, which activates protein kinase C. TRH is typical of a large class of regulatory molecules that act by similar mechanisms, and the information obtained on its detailed mechanism of action should be valuable to an overall understanding the control processes. Work will be performed using clonal lines of rat pituitary tumor cells (GH-cells) and normal rat pituitary cells in culture. The specific objectives are as follows. 1. The TRH receptor will be identified and purified, and antibodies against the receptor will be obtained. The following interdependent approaches will be used: cell lines with amplified receptor density will be selected; TRH receptors will be identified by photoaffinity labeling; receptors will be purified by conventional methods, including affinity chromatography; and anti-receptor antibodies will be raised. 2. The mechanism of cycling and regulation of TRH receptors will be established. Fluorescence microscopy will be used to monitor the intracellular localization of the ligand, using a fluorescent TRH derivative, and the receptors, using fluorescent probes to anti- receptor antibodies. The mechanism of homologous and heterologous TRH receptor regulation will be determined by measuring the effects of TRH, thyroid hormone, estrogen and cortisol on receptor concentration in intact and solubilized preparations, receptor mRNA levels, receptor phosphorylation state, and the fraction of cells with receptors. 3. The detailed mechanism of TRH action, particularly as relates to later phases of the response, will be determined. Individual cell responses to TRH will be monitored by fluorescence imaging methods designed to measure receptors, calcium response and protein kinase C activation. The role of voltage sensitive calcium channels in TRH response will be assessed, and the mechanism of long term TRH regulation of calcium channel activity will be established. 4. Once methods have been established for the biochemical identification TRH receptors and for monitoring TRH responses on single cells, these techniques will be applied to normal pituitary cells.